Responsible for up to 50% of cellular phosphoserine/threonine phosphatase activity, protein phosphatase 2A (PP2A) regulates almost all cell signaling pathways. PP2A comes as > 80 kinds of heterotrimers, consisting of a catalytic C subunit and one of many regulatory B subunits bound to an A? (90% abundant) or (10% abundant) A? subunit scaffold. Our premise is that protein phosphatase 2A (PP2A) using the A? scaffold is fundamentally important for controlling phenotypes of both normal and cancer cells. Moreover, study of polyomaviruses, which have repeatedly given novel insights into growth control, will be invaluable to understand A? function. Our studies of murine polyomavirus (MuPyV) ST/MT already demonstrate the importance of A? to survival, differentiation and transformation. Our sh/siRNA A? knockdowns of confirm its importance, even in the absence of virus, to pathways important in cancer. Both Akt and c-Src signaling are regulated by A?. In addition, human lung, breast and colon cancers show alterations in A?, suggesting that A?-PP2A-mediated signaling is relevant to cancer. Very little work has been done on A?, so there is a pressing need to study its function. In Aim 1 we will use broad-based technologies in a survey of functions and integrate these approaches to identify pathways altered by A? in normal and transformed cells. Expression analysis by RNA-seq, phosphoproteomic analysis, and NMR metabolomics will identify pathways specifically targeted by PP2A A?. Comparisons of controls with cells expressing MuPyV MT or ST will inform us whether the oncoproteins are inhibiting A? activity and/or redirecting it to new targets. Aim 2, focusing on A?, will determine the A?/ST structure. PP2A B subunits and other targets that bind A? will be determined. Genetic analysis of A? will uncover sequences responsible for its unique phenotype(s). In Aim 3 A? regulation of tyrosine kinase signaling will be examined to learn the mechanism of c-Src control and to determine how A? broadly controls tyrosine phosphorylation, perhaps via tyrosine phosphatases. ST mutants defective for A? binding will be identified to test the role of A? in controlling cell phenotype. Finally, we will confirm the role of A? in Her2/neu and MT tumorigenesis using conditional knockout technology. !